WO1997030178A2 - Diagnosing trinucleotide repeat diseases and genes involved therein - Google Patents

Diagnosing trinucleotide repeat diseases and genes involved therein Download PDF

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WO1997030178A2
WO1997030178A2 PCT/FR1997/000297 FR9700297W WO9730178A2 WO 1997030178 A2 WO1997030178 A2 WO 1997030178A2 FR 9700297 W FR9700297 W FR 9700297W WO 9730178 A2 WO9730178 A2 WO 9730178A2
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sequences
sequence
type
clone
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WO1997030178A3 (en
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Christian Neri
Howard M. Cann
Daniel Cohen
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Fondation Jean Dausset-Ceph
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Priority to JP9529061A priority Critical patent/JP2000505293A/en
Priority to EP97905219A priority patent/EP0894144A2/en
Publication of WO1997030178A2 publication Critical patent/WO1997030178A2/en
Publication of WO1997030178A3 publication Critical patent/WO1997030178A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the detection of human genes comprising repetitive triplet sequences CAG or CTG, as well as the use of these genes in the diagnosis and the possible treatment of certain hereditary neurological diseases.
  • CAG or CTG repeat triplet sequences constitutes a mutation involved in at least 6 human inherited neurological diseases (called “repeat triplet diseases”), in particular spinobulbar muscle atrophy (SBMA), myotonic dystrophy (MD), cerebro-spinal ataxia (SCAS) 1 and 3, den-ato-rubro-pallydoluysiane atrophy (DRPLA) and Huntington's disease (HD) (1).
  • SBMA spinobulbar muscle atrophy
  • MD myotonic dystrophy
  • SCAS cerebro-spinal ataxia
  • DRPLA den-ato-rubro-pallydoluysiane atrophy
  • HD Huntington's disease
  • CAG / CTG also called dynamic mutation
  • CAG / CTG repeats can occur in the non-coding (MD) or coding (eg SBMA and HD) regions of the transcripts. These transcripts are sometimes expressed in tissues other than the brain and when translated result in larger gene products carrying an abnormally elongated polyglutamine domain (2 to 4).
  • the foregoing demonstrates the considerable importance of repeated triplet diseases and the importance of their diagnosis and treatment.
  • the present invention is based on a systematic study of CAG / CTG repeats (which will be designated hereinafter by "[CAG] n", n being the number of repeats of the triplet), this study having been carried out with human cDNA libraries , these having undergone a first general screening, then the selected sequences having been selected on the basis of a certain number of criteria making it possible to ensure that the sequences in question belonged to new human genes and could, with great probability, to be implicated in diseases with repeated triplet. Finally, the selected sequences were the subject of a more complete study intended to allow their localization.
  • two sets of cDNAs from human brains were studied to analyze the presence of CAG repeats using hybridization of oligonucleotides on high density membranes.
  • the two libraries were obtained from mRNAs using the oligo-dT primer, one of the libraries being made up of fetal brain (FB) cDNA clones and the other library being made up of clones of Normalized newborn brain cDNA (NIB).
  • FB fetal brain
  • NBI Normalized newborn brain cDNA
  • human ESTs in databases have been analyzed for the presence of repeated triplets in cDNAs from human tissues other than the brain.
  • the present invention is based on the demonstration of certain sequences capable of being implicated in repeated triplet diseases obtained under hybridization conditions making it possible to select cDNAs which contain a number of sequences [CAG] greater than 9, which are more likely to be polymorphic in a normal population. 0
  • the present invention relates to a transcribed DNA sequence rich in repeated triplet CAG or CTG corresponding to sequences A to I of the table, as well as their normal and mutated alleles and the complementary sequences.
  • normal alleles is meant the alleles as they have been isolated or as they can be isolated from samples from normal individuals, the “mutated alleles” being the alleles of the genes carrying your sequences [CAG] n abnormally repeated.
  • sequences in question are for the first time identified as being part of a gene which may present a dynamic mutation, said genes not having , moreover, in themselves never been described.
  • sequences are identified in the table and can, if necessary, be re-isolated using the following primers: SEQ. ID 1 to 18, primers 1 to 1 8 corresponding, in pairs, to the sequences A to 1 mentioned above.
  • sequences thus highlighted can, first of all, be used within the framework of the diagnosis, and more exactly of a prognosis. Indeed, like a large number of diseases having a genetic support, the demonstration of the presence of a sequence presenting an abnormal number of repetitions of triplet [CAGjn cannot in itself ensure the occurrence of the disease, but must be interpreted according to a set of other information to allow either a very early diagnosis or possibly specific surveillance, especially in families at risk.
  • This diagnosis can be made by comparing the DNA sequence according to the patient's invention with a normal sequence to detect the presence of additional nucleotide repeats.
  • the present invention relates to a method for demonstrating the risks of the appearance of a trinucleotide recurrent disease in a patient, characterized in that the DNA sequence according to claim 1 of said patient is compared to a normal sequence to detect the presence of additional trinucleotide repeats.
  • a method for demonstrating the risks of the appearance of a trinucleotide recurrent disease in a patient characterized in that the DNA sequence according to claim 1 of said patient is compared to a normal sequence to detect the presence of additional trinucleotide repeats.
  • the most effective methods consist in carrying out an amplification operation, before highlighting the differences, by any suitable method, in particular the so-called "PCR" method, even if other methods can be used.
  • primers which can be used in the context of the methods according to the invention, mention should be made of the primers of SEQ . ID 1 to 1 S which constitute two by two of the pairs of primers for each of the sequences object of the invention.
  • the sequences according to the present invention are amplified and it is then possible, by comparison with a normal and / or standard sample, to demonstrate the presence of the supernumerary triplets and therefore the possibility of occurrence of a disease linked to this type of dynamic mutation. It is also interesting to note that these dynamically mutated diseases are known to be all the more severe and to occur the sooner the greater the number of repetitions.
  • the diagnostic method can allow not only a good prognosis for the onset of the disease, but also to assess the time when this disease will occur and / or its possible severity.
  • the present invention also relates to genes and their alleles which carry, at least in part, these sequences, said genes being, of course, directly involved in the onset of the disease.
  • the corresponding genes can be expressed in cells by known means in order to produce the corresponding proteins.
  • these proteins being involved in the disease, it is desired to reduce their quantity, either by blocking their expression by appropriate methods at the level of genes or regulatory elements, or by fixing or inactivating them, for example using receptor proteins acting as decoys. Again, these receptor or inactivated proteins can be generated in situ by expression of the corresponding DNA sequences.
  • proteins with abnormally large polyglutamic domains us av have abnormal aggregation properties, both with each other and with other proteins (7, 8)
  • the aggregates thus created are probably involved in the genesis of triplet repeat diseases, this is why it is also possible to provide a therapy in which the therapeutic agents would prevent aggregation, either by blocking the molecule as described above, or by attaching to the molecule to prevent approximation with other proteins.
  • sequences according to the present invention can be obtained thanks to the information appearing in the table and to the references which are given therein and by using the primer sequences which are described in the attached sequence identifiers.
  • the two libraries used are: a first library (5) containing 60,000 non-standard cDNAs of human fetal brain (FB clones) (Laboratory of Dr. Hans Lehrach, Max Plank Institute for Molecular Genetics, Berlin, Germany) subclones in the vector p-SPORT- 1 (Life Technologies, Inc., Gaithesburg,
  • the second library contains 40,128 normalized cDNAs for newborn brains (NIB clones) subcloned into a laf id (6) vector and which is part of the resources of the I MAGE consortium (Lawrence Livermore Lab ., Livermore, CA,) and the EST Merck WU program
  • clone 2.1 16 which is located on chromosome 3pl4 and possibly involved in ADCA II, the other highly polymorphic sequences [CAGjn previously described do not correspond to any known locus for hereditary neurological diseases.
  • This clone 2.1 16 was found to correspond to a transcript of 2227 base pairs highly expressed in skeletal muscles and weakly expressed in the brain according to the Northern Blot analysis. The repetition of the polymorphic CAG sequences is localized at the level of the 3 'untranslated region of the mRNA.
  • the RNA corresponding to clone 2.1 16 codes for a putative protein of 137 amino acids (nt 127 to 538) having no homology with proteins known in Genbank.
  • the complete sequence of the transcript of clone 2. 1 16 is represented by SEQ D 19.
  • IS sequence homologies with the IS in Genbank: nu mero access ESTs in Genbank

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Abstract

A transcribed DNA sequence with a high level of CAG or CTG repeat codons corresponding to sequences A to I on the table, and alleles and complementary sequences thereof, are disclosed. The sequences are particularly useful for diagnosing trinucleotide repeat diseases.

Description

DIAGNOSTIC DES MALADIES Λ REPETITION TRINUCLEOTIDIQUE ET GENES IMPLIQUES DANS CES MALADIESDIAGNOSIS OF TRINUCLEOTIDE REPETITIVE DISEASES AND GENES INVOLVED IN SUCH DISEASES
La présente invention concerne la mise en évidence de gènes humains comportant des séquences à triplet répété CAG ou CTG, ainsi que l'utilisation de ces gènes dans le diagnostic et l'éventuel traitement de certaines maladies neurologiques héréditaires.The present invention relates to the detection of human genes comprising repetitive triplet sequences CAG or CTG, as well as the use of these genes in the diagnosis and the possible treatment of certain hereditary neurological diseases.
L'expansion de séquences à triplet répété CAG ou CTG hautement polymorphiques et instables constitue une mutation impliquée dans au moins 6 maladies neurologiques héréditaires humaines (appelées "maladies à triplet répété ") , notamment l'atrophie musculaire spinobulbaire (SBMA), la dystrophie myotonique (MD), l'ataxie cérébro¬ spinale (SCAS) 1 et 3, l'atrophie den-ato-rubro-pallydoluysiane (DRPLA) et la maladie d'Huntington (HD) ( 1 ).The expansion of highly polymorphic and unstable CAG or CTG repeat triplet sequences constitutes a mutation involved in at least 6 human inherited neurological diseases (called "repeat triplet diseases"), in particular spinobulbar muscle atrophy (SBMA), myotonic dystrophy (MD), cerebro-spinal ataxia (SCAS) 1 and 3, den-ato-rubro-pallydoluysiane atrophy (DRPLA) and Huntington's disease (HD) (1).
L'expansion des CAG/CTG (appelée également mutation dynamique) dans chacune de ces maladies est associée à un phénomène d'anticipation, la taille des répétitions étant souvent corrélée de façon inverse avec l'âge de la survenue et/ou de la sévérité des symptômes.The expansion of CAG / CTG (also called dynamic mutation) in each of these diseases is associated with a phenomenon of anticipation, the size of the repetitions often being inversely correlated with the age of onset and / or severity symptoms.
L'expansion des répétitions CAG/CTG peut apparaître dans les régions non codantes (MD) ou codantes (par exemple SBMA et HD) des transcrits. Ces transcrits sont parfois exprimés dans les tissus autres que le cerveau et lorsqu'ils sont traduits conduisent à des produits de gènes plus grands portant un domaine polyglutamine anormalement allonge (2 à 4).The expansion of CAG / CTG repeats can occur in the non-coding (MD) or coding (eg SBMA and HD) regions of the transcripts. These transcripts are sometimes expressed in tissues other than the brain and when translated result in larger gene products carrying an abnormally elongated polyglutamine domain (2 to 4).
La mise en évidence d'expansion CAG/CTG dans l'ADN génomique de patients et/ou l'anticipation dans les familles à risque a suggéré que les mutations dynamiques sont impliquées dans SCA 2, SCA 4 et SCA 5, l'ataxie cérébrale dominante autosomale (ADCA de type 2) et la forme familiale du désordre affectif bipolaire ( BPAD) ou la schizophrénie. L'autisme et la démence familiale qui montrent des variabilités dans l'âge de la survenue ou dans la sévérité des symptômes pourraient également être causés par des mutations dynamiques.Evidence of CAG / CTG expansion in patient genomic DNA and / or anticipation in families at risk suggested that dynamic mutations are involved in SCA 2, SCA 4 and SCA 5, cerebral ataxia dominant autosomal (ADCA type 2) and the familial form of bipolar affective disorder (BPAD) or schizophrenia. Autism and familial dementia, which show variability in the age of onset or in the severity of symptoms, could also be caused by dynamic mutations.
Un assez grand nombre de maladies pourraient également avoir pour origine ou impliquer des mutations dynamiques :A fairly large number of diseases could also originate or involve dynamic mutations:
- la maladie de Parkinson,- Parkinson's disease,
- les paraplégies spastiques, - les ataxies cérébrospinale non répertoriées précédemment,- spastic paraplegias, - cerebrospinal ataxias not previously listed,
- les cataractes zonulaires, /30178- zonular cataracts, / 30178
- les tremblements incontrôlables,- uncontrollable tremors,
- les neuropathies amyloïdes familiales,- familial amyloid neuropathies,
- les arthrites granulomateuses familiales (maladie de Blau),- family granulomatous arthritis (Blau's disease),
- les microsomies hémifaciales, - certaines anémies et glaucomes,- hemifacial microsomies, - certain anemias and glaucomas,
- les désordres obsessionnels.- obsessive-compulsive disorder.
Ce q ui précède démon tre l 'importance considérable des maladies à triplet répété et l'importance de leur diagnostic et de leur traitement. La présente invention repose sur une étude systématique des répétitions CAG/CTG (qui seront ci-après désignées par "[CAG]n", n étant le nombre de répétitions du triplet), cette étude ayant été réalisée avec des banques d'ADNc humains, celles-ci ayant subi un premier criblage général, puis les séquences retenues ayant été sélectionnées sur la base d'un certain nombre de critères permettant de s'assurer que les séquences en cause appartenaient à de nouveaux gènes humains et pouvaient, avec une grande probabilité, être impliquées dans des maladies à triplet répété. Enfin, les séquences sélectionnées ont fait l'objet d'une étude plus complète destinée à permettre leur localisation. Dans le cadre de la présente invention, deux ensembles d'ADNc provenant de cerveaux humains ont été étudiés pour analyser la présence de répétitions CAG en utilisant l'hybridation d 'oligonucleotides su r des membranes à haute densité. Les deux librairies ont été obtenues à partir de ARNms à l'aide d'amorce oligo-dT, l'une des librairies étant constituée de clones d 'ADNc de cerveau foetal (FB) et l'autre librairie étant constituée de clones d'ADNc normalisés de cerveau de nouveau-né (NIB).The foregoing demonstrates the considerable importance of repeated triplet diseases and the importance of their diagnosis and treatment. The present invention is based on a systematic study of CAG / CTG repeats (which will be designated hereinafter by "[CAG] n", n being the number of repeats of the triplet), this study having been carried out with human cDNA libraries , these having undergone a first general screening, then the selected sequences having been selected on the basis of a certain number of criteria making it possible to ensure that the sequences in question belonged to new human genes and could, with great probability, to be implicated in diseases with repeated triplet. Finally, the selected sequences were the subject of a more complete study intended to allow their localization. In the context of the present invention, two sets of cDNAs from human brains were studied to analyze the presence of CAG repeats using hybridization of oligonucleotides on high density membranes. The two libraries were obtained from mRNAs using the oligo-dT primer, one of the libraries being made up of fetal brain (FB) cDNA clones and the other library being made up of clones of Normalized newborn brain cDNA (NIB).
De plus, les ESTs humains dans les banques de données ont été analysés pour détecter la présence de triplets répétés dans les ADNc de tissus humains autres que le cerveau. De façon générale, la présente invention repose sur la mise en évidence de certaines séquences susceptibles d'être impliquées dans les maladies à triplet répété obtenues dans des conditions d'hybridation permettant de sélectionner des ADNc qui contiennen t un nombre de séq uences [CAG] supérieur à 9, lesquelles sont plus susceptibles d'être polymorphes dans une population normale. 0In addition, human ESTs in databases have been analyzed for the presence of repeated triplets in cDNAs from human tissues other than the brain. In general, the present invention is based on the demonstration of certain sequences capable of being implicated in repeated triplet diseases obtained under hybridization conditions making it possible to select cDNAs which contain a number of sequences [CAG] greater than 9, which are more likely to be polymorphic in a normal population. 0
Les conditions complètes d'analyse et de sélection de ces différentes séquences ne seront pas détaillées ci-après, seuls seront fournis les éléments permettant de les localiser et de les rcisolcr grâce, notamment, aux amorces PCR qui seront décrites ci-après. Plus particulièrement, la présen te invention concerne une séquence d'ADN transcrit riche en triplet répété CAG ou CTG correspondant aux séquences A à I du tableau, ainsi que leurs alleles normaux et mutés et les séquences complémentaires.The complete conditions of analysis and selection of these different sequences will not be detailed below, only the elements making it possible to locate and to isolate them will be provided, in particular, thanks to the PCR primers which will be described below. More particularly, the present invention relates to a transcribed DNA sequence rich in repeated triplet CAG or CTG corresponding to sequences A to I of the table, as well as their normal and mutated alleles and the complementary sequences.
Par "alleles normaux" on entend désigner les alleles tels qu 'ils ont été isolés ou tels qu'il peuvent être isolés de prélèvements d'individus normaux, les "alleles mutés" étant les alleles des gènes portant tes séquences [CAG]n anormalement répétées.By "normal alleles" is meant the alleles as they have been isolated or as they can be isolated from samples from normal individuals, the "mutated alleles" being the alleles of the genes carrying your sequences [CAG] n abnormally repeated.
Il est important de noter que, bien que certaines de ces séquences soient, totalement ou en partie, publiques, les séquences en cause sont pour la première fois identifiées comme faisant partie d'un gène pouvant présenter une mutation dynamique, lesdits gènes n'ayant, par ailleurs, en eux-mêmes jamais été décrits.It is important to note that, although some of these sequences are, in whole or in part, public, the sequences in question are for the first time identified as being part of a gene which may present a dynamic mutation, said genes not having , moreover, in themselves never been described.
Les séquences ainsi mises en évidence présentent, notamment pour sept d 'entre elles, un pourcentage d'hétérozygotie (pourcentage HTZ) suffisamment important pour être impliquées très directement dans des maladies à triplet répété. Les séquences E et 1 qui présentent un très faible taux d'hétérozygotie (HTZ : 0,05) sont moins susceptibles d'être impliquées dans de telles maladies.The sequences thus highlighted present, in particular for seven of them, a percentage of heterozygosity (percentage HTZ) sufficiently large to be involved very directly in diseases with repeated triplets. Sequences E and 1 which have a very low rate of heterozygosity (HTZ: 0.05) are less likely to be involved in such diseases.
Bien entendu, comme cela est mentionné précédemment, ces séquences sont identifiées dans le tableau et pourront, si besoin est, être réisolées en utilisant les amorces suivantes : SEQ. ID 1 à 18, les amorces 1 à 1 8 correspondant, par paire, aux séquences A à 1 mentionnées précédemment.Of course, as mentioned previously, these sequences are identified in the table and can, if necessary, be re-isolated using the following primers: SEQ. ID 1 to 18, primers 1 to 1 8 corresponding, in pairs, to the sequences A to 1 mentioned above.
Les séquences ainsi mises en évidence peuvent, tout d'abord, être utilisées dans le cadre du diagnostic, et plus exactement d'un pronostic. En effet, comme un grand nombre de maladies ayant un support génétique, la mise en évidence de la présence d'une séquence présentant un nombre de répétitions anormal de triplet [CAGjn ne peut en elle-même assurer de la survenue de la maladie, mais doit être interprétée en fonction d'un ensemble d'autres informations pour permettre, soit un diagnostic très précoce, soit éventuellement une surveillance spécifique, surtout dans les familles à risque. Ce diagnostic peut être effectué en comparan t la séquence d'ADN selon l'invention du patient avec une séquence normale pour détecter la présence de répétitions tπnucléotidiqucs supplémentaires.The sequences thus highlighted can, first of all, be used within the framework of the diagnosis, and more exactly of a prognosis. Indeed, like a large number of diseases having a genetic support, the demonstration of the presence of a sequence presenting an abnormal number of repetitions of triplet [CAGjn cannot in itself ensure the occurrence of the disease, but must be interpreted according to a set of other information to allow either a very early diagnosis or possibly specific surveillance, especially in families at risk. This diagnosis can be made by comparing the DNA sequence according to the patient's invention with a normal sequence to detect the presence of additional nucleotide repeats.
Plus partic ulièrement, la présente invention concerne un procédé de mise en évidence des risques d 'apparition d'une maladie a répétition trinucléotidique chez un patient, caractérisé en ce qu 'on compare la séquence d'ADN selon la revendication 1 dudit patient à une séquence normale pour détecter la présence de répétitions trinucléotidiq ues supplémentaires. Bien en tendu, il existe un grand nombre de méthodes qui permettent la mise en évidence de triplets surnuméraires par rapport à des séquences normales, par exemple il est possible de mettre en évidence des différences de poids moléculaires en utilisant des gels et une méthode de type RFLP. Mais, les méthodes les plus efficaces consistent à pratiquer préalablement à la mise en évidence des différences une opération d'amplification, par toute méthode appropriée, notamment la méthode dite "PCR", même si d'autres méthodes sont utilisables.More particularly, the present invention relates to a method for demonstrating the risks of the appearance of a trinucleotide recurrent disease in a patient, characterized in that the DNA sequence according to claim 1 of said patient is compared to a normal sequence to detect the presence of additional trinucleotide repeats. Although in tension, there are a large number of methods which allow the highlighting of supernumerary triplets compared to normal sequences, for example it is possible to highlight differences in molecular weights using gels and a type method RFLP. However, the most effective methods consist in carrying out an amplification operation, before highlighting the differences, by any suitable method, in particular the so-called "PCR" method, even if other methods can be used.
Il n'est pas nécessaire ici de décrire en détail la méthode PCR, celle-ci permet d'amplifier de façon considérable une séquence spécifique comprise entre deux séquences appelées "amorces".It is not necessary here to describe in detail the PCR method, it makes it possible to considerably amplify a specific sequence comprised between two sequences called "primers".
Parmi les amorces utilisables dans le cadre des procédés selon l'invention, il faut citer les amorces des SEQ. ID 1 à 1 S qui consti tuent deux par deux des paires d'amorces pour chacune des séquences objet de l'inven tion. En utilisant les amorces décrites précédemment, on amplifie les séquences selon la présente invention et i l est alors possible, par comparaison avec un échantillon normal et/ou étalon, de mettre en évidence la présence des triplets surnuméraires et donc la possibilité de survenue d'une maladie liée à ce type de mutation dynamique. II est également intéressant de noter que ces maladies à mutation dynamique sont connues pour être d'autant plus sévères et survenir d'autant plus tôt qu'est important le nombre de répétitions. Dans ces conditions, la méthode diagnostic peut permettre, non seulement un bon pronostic de la survenue de la maladie, mais également d'évaluer le moment où cette maladie surviendra et/ou son éventuelle sévérité. La présente invention concerne également les gènes et leurs alleles qui portent, au moins en parties, ces séquences, lesdits gènes étant, bien entendu, impliqués directement dans la survenue de la maladieAmong the primers which can be used in the context of the methods according to the invention, mention should be made of the primers of SEQ . ID 1 to 1 S which constitute two by two of the pairs of primers for each of the sequences object of the invention. Using the primers described above, the sequences according to the present invention are amplified and it is then possible, by comparison with a normal and / or standard sample, to demonstrate the presence of the supernumerary triplets and therefore the possibility of occurrence of a disease linked to this type of dynamic mutation. It is also interesting to note that these dynamically mutated diseases are known to be all the more severe and to occur the sooner the greater the number of repetitions. Under these conditions, the diagnostic method can allow not only a good prognosis for the onset of the disease, but also to assess the time when this disease will occur and / or its possible severity. The present invention also relates to genes and their alleles which carry, at least in part, these sequences, said genes being, of course, directly involved in the onset of the disease.
Les gènes correspondant pourront être exprimés dans des cellules par des moyens connus afin de produire les protéines correspondantes.The corresponding genes can be expressed in cells by known means in order to produce the corresponding proteins.
Il est possible d'envisager l'utilisation desdues protéines dans certains kits de diagnostic par exemple.It is possible to consider the use of proteins in certain diagnostic kits for example.
De façon générale, ces protéines étant impliq uées dans la maladie, on souhaitera en diminuer la quantité, soit en bloquant leur expression par des méthodes appropriées au niveau des gènes ou des éléments de régulation, soit en les fixant ou en les inactivant, par exemple en utilisant des protéines réceptrices jouant le rôle de leurres. Là encore, ces protéines réceptrices ou inactivées pourront être générées m situ par expression des séquences d'ADN correspondantesIn general, these proteins being involved in the disease, it is desired to reduce their quantity, either by blocking their expression by appropriate methods at the level of genes or regulatory elements, or by fixing or inactivating them, for example using receptor proteins acting as decoys. Again, these receptor or inactivated proteins can be generated in situ by expression of the corresponding DNA sequences.
Il est également possible de prévoir la réalisation d 'anticorps monoclonaux correspondant à ces protéines afin d 'envisager le blocage desdites protéines lorsque cela est souhaité, l 'ensemble de ces opérations pouvant être réalisé directement in vivo par exemple, en utilisant des techniques de thérapie génique, en particulier en utilisant des vecteurs qui porteront les séquences d'expression des gènes.It is also possible to provide for the production of monoclonal antibodies corresponding to these proteins in order to envisage blocking said proteins when desired, all of these operations can be performed directly in vivo for example, using therapy techniques. gene, in particular using vectors that will carry the gene expression sequences.
On a mis en évidence le fait que les protéines présentant des domaines polyglutam es anormalement étend us av aient des propriétés d'aggrégation anormales, tant entre elles, qu'avec d'autres protéines (7, 8 ) Les aggrégats ainsi créés sont probablement impliques dans la genèse des maladies à triplet répète, c'est pourquoi il est également possible de prévoir une thérapie dans laquelle les agents thérapeutiques viendraient empêcher l'aggrégation, soit en bloquant la molécule comme décrit précédemment, soit en se fixant à la molécule pour empêcher le rapprochement avec d'autres protéines.It has been demonstrated that proteins with abnormally large polyglutamic domains us av have abnormal aggregation properties, both with each other and with other proteins (7, 8) The aggregates thus created are probably involved in the genesis of triplet repeat diseases, this is why it is also possible to provide a therapy in which the therapeutic agents would prevent aggregation, either by blocking the molecule as described above, or by attaching to the molecule to prevent approximation with other proteins.
On peut, dans le cadre de la thérapie, prévoir d'utiliser des variants complets ou délétés de ces protéines, normales ou non (quant au domaine [CAGjn).One can, within the framework of the therapy, plan to use complete or deleted variants of these proteins, normal or not (as for the domain [CAGjn).
Les séquences selon la présente invention peuvent être obtenues grâce aux informations figurant au tableau et aux références qui y sont données et en utilisant les séquences d'amorce qui sont décrues dans les identificateurs de séquence ci-joints. Les deux librairies utilisées sont : une première librairie (5 ) contenant 60 000 ADNc non normalisés de cerveau foetal humain (clones FB) (Laboratoire du Dr. Hans Lehrach, Max Plank Institute for Molecular Genetics, Berlin, Allemagne) sous- clones dans le vecteur p-SPORT- 1 (Life Technologies, Inc., Gaithesburg,The sequences according to the present invention can be obtained thanks to the information appearing in the table and to the references which are given therein and by using the primer sequences which are described in the attached sequence identifiers. The two libraries used are: a first library (5) containing 60,000 non-standard cDNAs of human fetal brain (FB clones) (Laboratory of Dr. Hans Lehrach, Max Plank Institute for Molecular Genetics, Berlin, Germany) subclones in the vector p-SPORT- 1 (Life Technologies, Inc., Gaithesburg,
MA, USA), et la seconde librairie contient 40 128 ADNc normalisés de cerveau de nouveau-né (clones NIB) sous-clonés dans un vecteur laf id (6) et qui est une partie des ressources du consortium I MAGE ( Lawrence Livermore Lab., Livermore, CA, ) et du programme EST Merck WUMA, USA), and the second library contains 40,128 normalized cDNAs for newborn brains (NIB clones) subcloned into a laf id (6) vector and which is part of the resources of the I MAGE consortium (Lawrence Livermore Lab ., Livermore, CA,) and the EST Merck WU program
(Washington University, St-Louis, LO, USA) .(Washington University, St-Louis, LO, USA).
D'autre part, un certain nombre d'autres informations ont été recueillies en utilisant Genbank Database (NCBl Bethesda, MA, USA).On the other hand, a number of other information was collected using the Genbank Database (NCBl Bethesda, MA, USA).
Les méthodes de sélection et d'analyse qui ont été mises en oeu\ re ne font pas en elles-mêmes partie de la présente invention puisque l'invention a essentiellement pour objet les séquences ainsi obtenues et leur utilisation, notamment, dans des méthodes de diagnostic ou des méthodes de traitement thérapeutique.The methods of selection and analysis which have been implemented do not in themselves form part of the present invention since the invention essentially relates to the sequences thus obtained and their use, in particular, in methods of diagnostic or therapeutic treatment methods.
Dans la présente analyse, on a retenu, non seulemen t les séquences polymorphiques [CAGjn parfaites, mais également les séquencesIn the present analysis, we have retained, not only the perfect polymorphic sequences [CAGjn, but also the sequences
[CAGjn complexes, c'est-à-dire celles qui sont ponctuées par des insertions de triplets ; en effet cette présence de triplets insérés s'observe notamment dans le cas de SCA 1 , alors que tel n'est pas le cas pour SBMA, MD et I ID.[CAGjn complexes, ie those which are punctuated by insertions of triplets; indeed, this presence of inserted triples is observed in particular in the case of SCA 1, while such is not the case for SBMA, MD and I ID.
L'étude présen te a montré que le polymorph isme [CAG j n apparaît lorsque la séquence (CAGjn contient plus de 9 copies CAG ( n > 9) ; c'est pourquoi, dans les sélections effectuées, les conditions de stringencc sont assez élevées, ce qui a permis de sélectionner rapidement un grand nombre de séquences [CAGj n dans lesquelles n est supérieur à 9. D'ailleurs, les séquences [CAGjn dans lesquelles n est inférieur à 10 et qui ont pu être testées dans le cad re de la présente inven tion on t montré u n polymorphisme nul.The present study showed that the polymorphism [CAG jn appears when the sequence (CAGjn contains more than 9 CAG copies (n> 9); this is why, in the selections made, the conditions of stringencc are quite high, which made it possible to quickly select a large number of sequences [CAGj n in which n is greater than 9. Moreover, the sequences [CAGjn in which n is less than 10 and which could be tested within the framework of the present invention we have shown a null polymorphism.
Par contre, il n'est pas possible de relever u ne corrélation directe entre la longueur des séquences [CAGj n et le degré de polymorphisme. 97/30178On the other hand, it is not possible to detect a direct correlation between the length of the sequences [CAGj n and the degree of polymorphism. 97/30178
77
D'autre part, les nouveaux clones sélectionnes a partir de librairies NIB sont distincts de ceux sélectionnés dans le groupe FB Ceci est en accord avec le fait que le cerveau humain exprime un grand nombre de gènes distincts à des stades de développement différents Enfin, des présentes observations il ressort que des séquencesOn the other hand, the new clones selected from NIB libraries are distinct from those selected from the FB group. This is in agreement with the fact that the human brain expresses a large number of distinct genes at different stages of development. present observations it appears that sequences
[CAGjn supérieures à 9 sont rares ( 1 pour 2 200 ) 1 pour 3 000) dans les ADNc humains représentatifs des régions 3' des ARNm.[CAGjn greater than 9 are rare (1 in 2,200) 1 in 3,000) in human cDNAs representative of the 3 ′ regions of the mRNAs.
Bien que dans l'état actuel de la présente invention , les séquences qui constituent l'objet de l'invention n'aient pas été reliées directement à des affections neurologiques héréditaires, la présence d'extensions anormales CAG peut être reliée avec le risque de survenue d'une maladie neurologique, en particulier lorsqu 'il y a une composante, familiale.Although in the present state of the present invention, the sequences which constitute the subject of the invention have not been directly linked to hereditary neurological disorders, the presence of abnormal CAG extensions may be linked with the risk of occurrence of a neurological disease, especially when there is a family component.
Sauf pour le clone 2.1 16 qui est localisé sur le chromosome 3pl4 et peut-être impliqué dans ADCA II, les autres séquences hautement polymorphiques [CAGjn décrues précédemment ne correspondent à aucun locus connu pour des maladies neurologiques héréditaires. Il a été trouvé que ce clone 2.1 16 correspondait à un transcrit de 2 227 paires de base hautement exprimé dans les muscles squelettiques et faiblement exprimé dans le cerveau d'après l'analyse Northern Blot. La répétition des séquences CAG polymorphiques est localisée au niveau de la région 3 ' non traduite de l'ARNm. L'ARN correspondant au clone 2.1 16 code pour une protéine putative de 137 acides aminés (nt 127 à 538) ne présentant aucu ne homologie avec des protéines connues dans Genbank La séquence complète du transcrit du clone 2. 1 16 est représentée par SEQ D 19.Except for clone 2.1 16 which is located on chromosome 3pl4 and possibly involved in ADCA II, the other highly polymorphic sequences [CAGjn previously described do not correspond to any known locus for hereditary neurological diseases. This clone 2.1 16 was found to correspond to a transcript of 2227 base pairs highly expressed in skeletal muscles and weakly expressed in the brain according to the Northern Blot analysis. The repetition of the polymorphic CAG sequences is localized at the level of the 3 'untranslated region of the mRNA. The RNA corresponding to clone 2.1 16 codes for a putative protein of 137 amino acids (nt 127 to 538) having no homology with proteins known in Genbank. The complete sequence of the transcript of clone 2. 1 16 is represented by SEQ D 19.
Néanmoins, il existe un assez grand nombre de maladies neurologiques qui actuellement n'ont pas été localisées et trouveront probablement à être reliées avec les séquences selon la présente invention Parmi les éléments additionnels qui ont été pris en compte pour étudier la pertinence des séquences retenues figure l'existence éventuelle d'un cadre de lecture ouvert (ORF) dans lequel les séquences [CAGjn codent pour une extension polyglutamine.Nevertheless, there are a fairly large number of neurological diseases which currently have not been localized and will probably find a link with the sequences according to the present invention. Among the additional elements which have been taken into account in order to study the relevance of the sequences retained. the possible existence of an open reading frame (ORF) in which the sequences [CAGjn code for a polyglutamine extension.
Enfin, la présente étude suggère que la fréquence d u polymorphisme [CAGjn est un événement rare dans les ADNc humains représentatifs des régions 3' des ARNm. TABLEAUFinally, the present study suggests that the frequency of polymorphism [CAGjn is a rare event in human cDNAs representative of the 3 'regions of mRNAs. BOARD
ICAGln polymoφhiques : caractéristiquesPolymorphic ICAGln: characteristics
Figure imgf000010_0001
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000011_0001
Clone : Nom du clone d'ADNc (nom CEPH) - Mode de sélection - Répétition 5"-3'Clone: Name of the cDNA clone (CEPH name) - Selection mode - Repeat 5 " -3 '
EST : Homologies de séquence avec les EST dans Genbank : nu méro d'accès des ESTs dans GenbankIS: sequence homologies with the IS in Genbank: nu mero access ESTs in Genbank
YAC(s) posltifis) : (Noms du CEPH) pour la séquence testéeYAC (s) posltifis): (Names of CEPH) for the sequence tested
STS+/STS- : YAC posi if /négatif pour d'au tres STS(s) Alu target : YAC non utilisé comme sonde Alu Alu probe : YAC utilisé comme sonde AluSTS + / STS-: YAC positive / negative for other STS (s) Alu target: YAC not used as Alu probe Alu probe: YAC used as Alu probe
Localisation : Localisation de la séquence testée dans les chromosomes humains (déduction d'après les YACs positifs) Location: Location of the sequence tested in human chromosomes (net of after positive YAC)
LISTE DE SEQUENCESLIST OF SEQUENCES
( 1 ) INFORMATIONS GENERALES (i) DEPOSANT : (A) NOM : FONDATION JEAN DAUSSET - CENTRE D'ETUDE DU(1) GENERAL INFORMATION (i) DEPOSITOR: (A) NAME: JEAN DAUSSET FOUNDATION - STUDY CENTER
POLYMORPHISME HUMAIN (CEPH).HUMAN POLYMORPHISM (CEPH).
(B) RUE : 27 Rue Juliette Dodu(B) STREET: 27 Rue Juliette Dodu
(C) VILLE : PARIS(C) CITY: PARIS
(D) PAYS : FRANCE (E) CODE POSTAL : 75010(D) COUNTRY: FRANCE (E) POSTAL CODE: 75010
(ii) TITRE DE L'INVENTION : DIAGNOSTIC DES MALADIES A REPETITION(ii) TITLE OF THE INVENTION: DIAGNOSIS OF REPETITIVE DISEASES
TRINUCLEOTIDIQUE ET GENES IMPLIQUES DANS CES MALADIESTRINUCLEOTIDE AND GENES INVOLVED IN THESE DISEASES
(iii) NOMBRE DE SEQUENCES : 18(iii) NUMBER OF SEQUENCES: 18
(iv) FORME DECHIFFRABLE PAR ORDINATEUR :(iv) COMPUTER-DETACHABLE FORM:
(A) TYPE DE SUPPORT : Floppy disk (B) ORDINATEUR : MACINTOSH APPLE(A) TYPE OF MEDIUM: Floppy disk (B) COMPUTER: MACINTOSH APPLE
(C) SYSTEM E D'EXPLOITATION : MAC-OS SYSTEME 7(C) OPERATING SYSTEM E: MAC-OS SYSTEM 7
( D) LOGICIEL : WORDPERFECT(D) SOFTWARE: WORDPERFECT
(v) DONNEES DE LA DEMANDE ACTUELLE : NUMERO DE LA DEMANDE :(v) CURRENT REQUEST DATA: REQUEST NUMBER:
(2) INFORMATIONS POUR LA SEQID NO 1 :(2) INFORMATION FOR SEQID NO 1:
. (i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR : 15 nucléotides. (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 nucleotides
(B) TYPE : nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION : linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc (iii) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEU R DE LA MOLECULE :(ii) TYPE OF MOLECULE: cDNA (iii) CHARACTERISTICS OF THE CARRIER CELL CLONE OF THE MOLECULE:
(A) TYPE DE CELLULE : bactérie(A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : 2.1 16 (C) ORIGINE : Max Plank Institute for Molecular Genctics(B) NAME OF THE cDNA CLONE IN THE APPLICANT: 2.1 16 (C) ORIGIN: Max Plank Institute for Molecular Genctics
Laboratory of Dr Hans Lehrach, Berlin, Allemagne. (D) NOM DU CLONE DANS LA BANQUE D'ORIGINE :ICRFp507E07266Laboratory of Dr Hans Lehrach, Berlin, Germany. (D) NAME OF THE CLONE IN THE BANK OF ORIGIN: ICRFp507E07266
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
CCAGCCTCAG GTAGC 15CCAGCCTCAG GTAGC 15
(2) INFORMATIONS POUR LA SEQID NO 2 :(2) INFORMATION FOR SEQID NO 2:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 22 nucléotides(A) LENGTH: 22 nucleotides
(B) TYPE : nucléotide(B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION : linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc(ii) TYPE OF MOLECULE: cDNA
( i ii ) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEU R DE LA MOLECULE : (A) TYPE DE CELLULE : bactérie(i ii) CHARACTERISTICS OF THE MOLECULAR CARRIER CELL CLONE: (A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : 2.1 16(B) NAME OF THE cDNA CLONE IN THE APPLICANT: 2.1 16
(C) ORIGINE : Max Plank Institute for Molecular Genetics,(C) ORIGIN: Max Plank Institute for Molecular Genetics,
Laboratory of Dr Hans Lehrach, Berlin, AllemagneLaboratory of Dr Hans Lehrach, Berlin, Germany
(D) NOM DU CLONE DANS LA BANQUE D'ORIGINE :ICRFp507E07266(D) NAME OF THE CLONE IN THE BANK OF ORIGIN: ICRFp507E07266
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
GTACTGAGGG CTTTTTAGAT TC 22 (2) INFORMATIONS POUR LA SEQ ID NO 3 :GTACTGAGGG CTTTTTAGAT TC 22 (2) INFORMATION FOR SEQ ID NO 3:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 18 nucléotides(A) LENGTH: 18 nucleotides
(B) TYPE : nucléotidc(B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple(C) NUMBER OF STRANDS: single
(D) CONFIG URATION : linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc(ii) TYPE OF MOLECULE: cDNA
(iii) CARACTERISTIQUES DU CLONE CELLU LAIRE PORTEUR DE LA MOLECULE :(iii) CHARACTERISTICS OF THE MOLECULAR CARRYING CLONE CELL:
(A) TYPE DE CELLULE : bactérie(A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : 2.1 19 (C) ORIGINE : Max Plank Institute for Molecular Genetics,(B) NAME OF THE cDNA CLONE IN THE APPLICANT: 2.1 19 (C) ORIGIN: Max Plank Institute for Molecular Genetics,
Laboratory of Dr Hans Lehrach, Berlin, Allemagne (D) NOM DU CLONE DANS LA BANQUE D'ORIGINE :ICRFp507J 10268Laboratory of Dr Hans Lehrach, Berlin, Germany (D) NAME OF CLONE IN BANK OF ORIGIN: ICRFp507J 10268
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
TCATGCAGCA GAAAACAG 18TCATGCAGCA GAAAACAG 18
(2) INFORMATIONS POUR LA SEQID NO 4 :(2) INFORMATION FOR SEQID NO 4:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 18 nucléotides(A) LENGTH: 18 nucleotides
(B) TYPE : nucléotidc(B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION : linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc(ii) TYPE OF MOLECULE: cDNA
(iii) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEUR DE LA MOLECULE : (A) TYPE DE CELLULE : bactérie(iii) CHARACTERISTICS OF THE MOLECULE-CARRYING CELL CLONE: (A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT :(B) NAME OF THE cDNA CLONE IN THE APPLICANT:
(C) ORIGINE : Max Plank Institute for Moleculai Genetics,(C) ORIGIN: Max Plank Institute for Moleculai Genetics,
Laboratory of Dr Hans Lehrach, Berlin, Allemagne (D) NOM DU CLONE DANS LA BANQUE D'ORIGINE .!CRFp507J 10268Laboratory of Dr Hans Lehrach, Berlin, Germany (D) NAME OF THE CLONE IN THE BANK OF ORIGIN.! CRFp507J 10268
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
AAAGGGGAGA CCAATTTG 18AAAGGGGAGA CCAATTTG 18
(2) INFORMATIONS POUR LA SEQID NO 5 :(2) INFORMATION FOR SEQID NO 5:
(i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR : 18 nucléotides (B) TYPE : nucléotide(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 nucleotides (B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION : linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc(ii) TYPE OF MOLECULE: cDNA
(iii) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEU R DE LA MOLECULE :(iii) CHARACTERISTICS OF THE CARRIER CELL CLONE OF THE MOLECULE:
(A) TYPE DE CELLULE : bactérie(A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : 2.70 (C) ORIGINE : Max Plank Institute for Molecular Genetics,(B) NAME OF THE cDNA CLONE IN THE APPLICANT: 2.70 (C) ORIGIN: Max Plank Institute for Molecular Genetics,
Laboratory of Dr Hans Lehrach, Berlin, Allemagne (D) NOM DU CLONE DANS LA BANQUE DORIGINE.CRFp507K242 1 2Laboratory of Dr Hans Lehrach, Berlin, Germany (D) NAME OF THE CLONE IN THE DORIGIN BANK.CRFp507K242 1 2
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
GCACAGTCCT CACTAAAC 18GCACAGTCCT CACTAAAC 18
(2) INFORMATIONS POUR LA SEQ ID NO 6 : (i) CARACTERISTIQUES DE LA SEQUENCE(2) INFORMATION FOR SEQ ID NO 6: (i) CHARACTERISTICS OF THE SEQUENCE
(A) LONGUEUR 19 nucléotides(A) LENGTH 19 nucleotides
(B) TYPE : nucleotide(B) TYPE: nucleotide
(C) NOMBRE DE BRIN simple (D) CONFIGURATION : linéaire(C) NUMBER of single STRANDS (D) CONFIGURATION: linear
(n) TYPE DE MOLECULE ADNc(n) TYPE OF cDNA MOLECULE
(m) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEUR DE LA MOLECULE • (m) CHARACTERISTICS OF THE MOLECULE-CARRYING CELL CLONE •
(A) TYPE DE CELLULE . bactérie(A) TYPE OF CELL. bacterium
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT 2 70(B) NAME OF THE cDNA CLONE IN THE DEPOSITOR 2 70
(C) ORIGINE Max Plank Institute for Molecular Genetics,(C) ORIGIN Max Plank Institute for Molecular Genetics,
Laboratory of Dr Hans Lehrach, Berlin, Allemagne (D) NOM DU CLONE DANS LA BANQUE D'ORlGiNLICRFp507K242 l 2Laboratory of Dr Hans Lehrach, Berlin, Germany (D) NAME OF THE CLONE IN THE BANK OF ORlGiNLICRFp507K242 l 2
(iv) DESCRIPTION DE LA SEQUENCE(iv) DESCRIPTION OF THE SEQUENCE
GGACATTGGC TTCAACTTC 19GGACATTGGC TTCAACTTC 19
(2 ) INFORMAT IONS TOUR LA SEQID NO 7(2) INFORMATION ON THE SEQID NO 7
(i) CARACTERISTIQUES DE LA SEQUENCE (A) LONGUEUR 16 nucléotides (B) TYPE nucleotide(i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 16 nucleotides (B) TYPE nucleotide
(C) NOMBRE DE BRIN . simple(C) NUMBER OF STRANDS. simple
(D) CONFIGURATION • linéaire(D) CONFIGURATION • linear
(n) TYPE DE MOLECULE . ADNc(n) TYPE OF MOLECULE. CDNA
(ni) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEUR DE LA MOLECULE(ni) CHARACTERISTICS OF THE MOLECULE-CARRYING CELL CLONE
(A) TYPE DE CELLULE bactérie ( B) NOM DU CLONE D'ADNc CHEZ LE DEPOSAN1 2 1 (C) ORIGINE Max Plank Institute for Molecular Genetics,(A) TYPE OF BACTERIA CELL (B) NAME OF THE cDNA CLONE IN THE DEPOSAN1 2 1 (C) ORIGIN Max Plank Institute for Molecular Genetics,
Laboratory of Dr Hans Lehrach, Berlin, Allemagne (D) NOM DU CLONE DANS LA BANQUE D'ORIGINE . ICREp50710522 (iv) DESCRIPTION DE LA SEQUFNCE • Laboratory of Dr Hans Lehrach, Berlin, Germany (D) NAME OF THE CLONE IN THE BANK OF ORIGIN. ICREp50710522 (iv) DESCRIPTION OF THE SEQUFNCE •
CAGGTGCAGC GTCAAA 16CAGGTGCAGC GTCAAA 16
(2) INFORMATIONS POUR LA SEQID NO 8 :(2) INFORMATION FOR SEQID NO 8:
(i) CARACTERISTIQUES DE LA SEQUENCE(i) CHARACTERISTICS OF THE SEQUENCE
(A) LONGUEUR : 16 nucléotides(A) LENGTH: 16 nucleotides
(B) TYPE : nucleotide (C) NOMBRE DE BRIN : simple(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single
(D) CONFIGURATION : linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc(ii) TYPE OF MOLECULE: cDNA
(m) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEU R DE LA(m) CHARACTERISTICS OF THE CARRIER CELL CLONE OF THE
MOLECULEMOLECULE
(A) TYPE DE CELLULE : bactérie(A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : 2.81(B) NAME OF THE cDNA CLONE IN THE APPLICANT: 2.81
(C) ORIGINE : Max Plank Institute for Molecular Genetics, Laboratory of Dr Hans Lehrach, Berlin, Allemagne(C) ORIGIN: Max Plank Institute for Molecular Genetics, Laboratory of Dr Hans Lehrach, Berlin, Germany
(D) NOM DU CLONE DANS LA BANQUE D'ORIGINE :!CRFp507!05222(D) NAME OF THE CLONE IN THE BANK OF ORIGIN:! CRFp507! 05222
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
GGAGGAGGTG TCACAG 16GGAGGAGGTG TCACAG 16
(2) INFORMATIONS POUR LA SEQID NO 9 :(2) INFORMATION FOR SEQID NO 9:
(i) CARACTERISTIQUES DE LA SEQUENCE. (A) LONGUEUR : 19 nucléotides(i) CHARACTERISTICS OF THE SEQUENCE. (A) LENGTH: 19 nucleotides
(B) TYPE : nucleotide(B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION : linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc (iii ) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEU R DE LA MOLECULE :(ii) TYPE OF MOLECULE: cDNA (iii) CHARACTERISTICS OF THE CARRIER CELL CLONE OF THE MOLECULE:
(A) TYPE DE CELLULE : bactérie(A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : i.S(B) NAME OF THE cDNA CLONE IN THE APPLICANT: i.S
(C) ORIGINE : Réseau I.M.A.G.E., Lawrence Livermore National(C) ORIGIN: I.M.A.G.E. Network, Lawrence Livermore National
Laboratories, Livermore, CA, USA.Laboratories, Livermore, CA, USA.
(D) NOM DU CLONE DANS LA BANQUE D'ORIGINE : 30262(D) NAME OF CLONE IN BANK OF ORIGIN: 30262
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
GATAAAAGGA AGGGAAAAG 19GATAAAAGGA AGGGAAAAG 19
(2) INFORMATIONS POUR LA SEQID NO 10:(2) INFORMATION FOR SEQID NO 10:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 17 nucléotides(A) LENGTH: 17 nucleotides
( B) TYPE : nucleotide(B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION : linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc(ii) TYPE OF MOLECULE: cDNA
(iii ) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEU R DE LA MOLECULE : (A) TYPE DE CELLULE : bactérie(iii) CHARACTERISTICS OF THE MOLECULAR CARRIER CELL CLONE: (A) CELL TYPE: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : i.8(B) NAME OF THE cDNA CLONE IN THE APPLICANT: i.8
(C) ORIGINE : Réseau I.M.A.G.E., Lawrence Livermore National(C) ORIGIN: I.M.A.G.E. Network, Lawrence Livermore National
Laboratories, Livermore, CA, USA.Laboratories, Livermore, CA, USA.
(D) NOM DU CLONE DANS LA BANQUE D'ORIGINE : 30262(D) NAME OF CLONE IN BANK OF ORIGIN: 30262
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
GCAACACTCA GAAATGG 17 (2) INFORMATIONS POUR LA SEQ ID NO 1 1 :GCAACACTCA GAAATGG 17 (2) INFORMATION FOR SEQ ID NO 1 1:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 19 nucléotides(A) LENGTH: 19 nucleotides
(B) TYPE : nucléotidc(B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION : linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc(ii) TYPE OF MOLECULE: cDNA
(iii) CARACTERISTIQUES DU CLONE CELLULAI RE PORTEU R DE LA MOLECULE :(iii) CHARACTERISTICS OF THE CELLULAI CLONE REPORTED FROM THE MOLECULE:
(A) TYPE DE CELLULE : bactérie(A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : i. 180 (C) ORIGINE : Réseau I.M.A.G.E., Lawrence Livermore National(B) NAME OF THE cDNA CLONE IN THE APPLICANT: i. 180 (C) ORIGIN: I.M.A.G.E. Network, Lawrence Livermore National
Laboratories, Ljvermore, CA, USA. (D) NOM DU CLONE DANS LA BANQUE D'ORIGINE : 153781 ou 162267Laboratories, Ljvermore, CA, USA. (D) NAME OF THE CLONE IN THE BANK OF ORIGIN: 153781 or 162267
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
AAGTCAGAGT TACTCTTGC 19AAGTCAGAGT TACTCTTGC 19
(2) INFORMATIONS POUR LA SEQ ID NO 1 2 :(2) INFORMATION FOR SEQ ID NO 1 2:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 17 nucléotides(A) LENGTH: 17 nucleotides
(B) TYPE : nucleotide(B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple (D) CONFIGURATION : linéaire(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc (ni) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEUR DE LA MOLECULE(ii) TYPE OF MOLECULE: cDNA (ni) CHARACTERISTICS OF THE MOLECULE-CARRYING CELL CLONE
(A) TYPE DE CELLULE : bactérie(A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT i 180 (C) ORIGINE : Reseau I M A.G.E , I-aw rence Livermor e National(B) NAME OF THE cDNA CLONE IN THE APPLICANT i 180 (C) ORIGIN: Reseau I M A.G.E, I-aw rence Livermor e National
Laboratories, Livermore, CA, USA. (D) NOM DU CLONE DANS LA BANQUE D'ORIGINE 153781 ou 162267Laboratories, Livermore, CA, USA. (D) NAME OF THE CLONE IN THE BANK OF ORIGIN 153781 or 162267
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
GAGTGAAGTT CAGGAGG 17GAGTGAAGTT CAGGAGG 17
(2) INFORMATIONS POUR LA SEQID NO 13 :(2) INFORMATION FOR SEQID NO 13:
(i) CARACTERISTIQUES DE LA SEQUENCE(i) CHARACTERISTICS OF THE SEQUENCE
(A) LONGUEUR : 19 nucléotides(A) LENGTH: 19 nucleotides
(B) TYPE . nucleotide(B) TYPE. nucleotide
(C) NOMBRE DE BRIN : simple (D) CONFIGURATION : linéaire(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear
(u) TYPE DE MOLECULE ADNc(u) TYPE OF cDNA MOLECULE
( m) CARACTERISTIQUES DU CLONE CELLULAI RE PORTEU R DE L\ MOLECULE .(m) CHARACTERISTICS OF THE CELLULAI CLONE RE CARRIER OF THE MOLECULE.
(A) TYPE DE CELLULE : bactérie(A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT . 1.181(B) NAME OF THE cDNA CLONE IN THE APPLICANT. 1.181
(C) ORIGINE : Réseau I. M. A.G.E., Lawrence Livermore National(C) ORIGIN: Network I. M. A.G.E., Lawrence Livermore National
Laboratories, Livermore, CA, USA. (D) NOM DU CLONE DANS LA BANQUE D'ORIGINE 1 14128Laboratories, Livermore, CA, USA. (D) NAME OF THE CLONE IN THE BANK OF ORIGIN 1 14128
(iv) DESCRIPTION DE LA SEQUENCE(iv) DESCRIPTION OF THE SEQUENCE
GGACAAAGCT ACATGTCAG 19 19GGACAAAGCT ACATGTCAG 19 19
(2) INFORMATIONS POUR LA SEQID NO 14 :(2) INFORMATION FOR SEQID NO 14:
(i) CARACTERISTIQUES DE LA SEQUENCE (A) LONGUEUR : 16 nucléotides (B) TYPE : nucleotide(i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 16 nucleotides (B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION : linéaire(D) CONFIGURATION: linear
(n) TYPE DE MOLECULE : ADNc(n) TYPE OF MOLECULE: cDNA
(ni) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEUR DE LA MOLECULE :(ni) CHARACTERISTICS OF THE MOLECULE-CARRYING CELL CLONE:
(A) TYPE DE CELLULE : bactérie(A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : 1.181 (C) ORIGINE : Réseau LM.A.G.E , Lawrence Livermore National(B) NAME OF THE cDNA CLONE IN THE APPLICANT: 1.181 (C) ORIGIN: LM.A.G.E Network, Lawrence Livermore National
Laboratories, Livermore, CA, USA. (D) NOM DU CLONE DANS LA BANQUE D'ORIGINE . 1 14128Laboratories, Livermore, CA, USA. (D) NAME OF THE CLONE IN THE BANK OF ORIGIN. 1 14128
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
GGTGAGTGTC CTTCTG 16GGTGAGTGTC CTTCTG 16
(2) INFORMATIONS POUR LA SEQID NO 15 .(2) INFORMATION FOR SEQID NO 15.
(i) CARACTERISTIQUES DE LA SEQUENCE(i) CHARACTERISTICS OF THE SEQUENCE
(A) LONGUEUR : 15 nucléotides(A) LENGTH: 15 nucleotides
(B) TYPE : nucleotide(B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION : linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc(ii) TYPE OF MOLECULE: cDNA
(iii ) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEUR DE LA MOLECULE : (A) TYPE DE CELLULE : bactérie(iii) CHARACTERISTICS OF THE MOLECULE-CARRYING CELL CLONE: (A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : i 182 (C) ORIGINE : Réseau LM.A.G.E., Lau rence Livermore National(B) NAME OF THE cDNA CLONE IN THE APPLICANT: i 182 (C) ORIGIN: LM.AGE Network, Lau rence Livermore National
Laboratories, Livermore, CA, USA.Laboratories, Livermore, CA, USA.
(D) NOM DU CLONE DANS LA BANQUE D'ORIGINE : 67444(D) NAME OF CLONE IN BANK OF ORIGIN: 67444
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
GGGCTAAGGG GAAAG 15GGGCTAAGGG GAAAG 15
(2) INFORMATIONS POUR LA SEQ ID NO 16 :(2) INFORMATION FOR SEQ ID NO 16:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 15 nucléotides(A) LENGTH: 15 nucleotides
(B) TYPE : nucleotide(B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple (D) CONFIGURATION : linéaire(C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc(ii) TYPE OF MOLECULE: cDNA
(iii ) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEU R DE LA MOLECULE :(iii) CHARACTERISTICS OF THE CARRIER CELL CLONE OF THE MOLECULE:
(A) TYPE DE CELLULE : bactérie(A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : 1.182(B) NAME OF THE cDNA CLONE IN THE APPLICANT: 1.182
(C) ORIGINE : Réseau LM.A.G. E., Lawrence Livermore National(C) ORIGIN: LM.A.G. network E., Lawrence Livermore National
Laboratories, Livermore, CA, USA. (D) NOM DU CLONE DANS LA BANQUE D'ORIGINE : 67444Laboratories, Livermore, CA, USA. (D) NAME OF CLONE IN BANK OF ORIGIN: 67444
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
CTTGGTGGGC AAGTG 15CTTGGTGGGC AAGTG 15
(2) INFORMATIONS POUR LA SEQ ID NO 17 :(2) INFORMATION FOR SEQ ID NO 17:
(i) CARACTERISTIQUES DE LA SEQUENCE: (A) LONGUEUR : 18 nucléotides (B) TYPE : nucleotide(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 nucleotides (B) TYPE: nucleotide
(C) NOMBRE DE BRIN : simple(C) NUMBER OF STRANDS: single
(D) CONFIGURATION : linéaire (ii) TYPE DE MOLECULE : ADNc(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA
(iii ) CARACTERISTIQUES DU CLONE CELLULAIRE PORTEUR DE LA MOLECULE : (A) TYPE DE CELLULE : bactérie(iii) CHARACTERISTICS OF THE MOLECULE-CARRYING CELL CLONE: (A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : 2.46(B) NAME OF THE cDNA CLONE IN THE APPLICANT: 2.46
(C) ORIGINE : Max Plank Institute for molecular Genetics,(C) ORIGIN: Max Plank Institute for molecular Genetics,
Laboratory of Dr Hans Lehrach, Berlin, Allemagne.Laboratory of Dr Hans Lehrach, Berlin, Germany.
(D) NOM DU CLONE DANS LA BANQUE D'ORIGINE :ICRFp507E06174(D) NAME OF THE CLONE IN THE BANK OF ORIGIN: ICRFp507E06174
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
TTTTTACTCG CGGCGGTG 18TTTTTACTCG CGGCGGTG 18
(2) INFORMATIONS POUR LA SEQ ID NO 1 8 :(2) INFORMATION FOR SEQ ID NO 1 8:
(i) CARACTERISTIQUES DE LA SEQUENCE:(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR : 25 nucléotides(A) LENGTH: 25 nucleotides
(B) TYPE : nucleotide (C) NOMBRE DE BRIN : simple(B) TYPE: nucleotide (C) NUMBER OF STRANDS: single
( D) CONFIGURATION : linéaire(D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE : ADNc(ii) TYPE OF MOLECULE: cDNA
( iii ) CARACTERISTIQUES DU CLONE CELLULAIR E PORTEU R DE LA(iii) CHARACTERISTICS OF THE CELLULAR CLONE AND CARRIER OF THE
MOLECULE :MOLECULE :
(A) TYPE DE CELLULE : bactérie(A) TYPE OF CELL: bacteria
(B) NOM DU CLONE D'ADNc CHEZ LE DEPOSANT : 2.46(B) NAME OF THE cDNA CLONE IN THE APPLICANT: 2.46
(C) ORIGINE : Max Plank Institute for molecular Genetics, Laboratory of Dr Hans Lehrach, Berlin, Allemagne.(C) ORIGIN: Max Plank Institute for molecular Genetics, Laboratory of Dr Hans Lehrach, Berlin, Germany.
(D) NOM DU CLONE DANS LA BANQUE D'ORIGINE : lCRfρ507E06 l 74(D) NAME OF THE CLONE IN THE BANK OF ORIGIN: lCRfρ507E06 l 74
(iv) DESCRIPTION DE LA SEQUENCE :(iv) DESCRIPTION OF THE SEQUENCE:
CAGGATAATC AAGAATGAAG TTAAG 25 ~)1CAGGATAATC AAGAATGAAG TTAAG 25 ~) 1
(2) INFORMATIONS POUR LA SEQ ID NO: 19(2) INFORMATION FOR SEQ ID NO: 19
i) CARACTERISTIQUES DE LA SEQUENCEi) CHARACTERISTICS OF THE SEQUENCE
(A) LONGUEUR: 2227 nucléotides(A) LENGTH: 2227 nucleotides
(B) TYPE: nucleotide(B) TYPE: nucleotide
(C) NOMBRE DE BRINS, simple(C) NUMBER OF STRANDS, single
(D) CONFIGURATION- linéaire(D) CONFIGURATION- linear
(ii) TYPE DE MOLECULE. ADNc(ii) TYPE OF MOLECULE. CDNA
(îx) CARACTERISTIQUE(îx) CHARACTERISTIC
(A) NOM/CLE: 2.116(A) NAME / KEY: 2.116
(B) EMPLACEMENTxhromosome 3pl4(B) LOCATIONxhromosome 3pl4
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO.19 gccggaagtggggtg gaagccccggtgctggtgcggcgggggactgcggggccagccec aggtagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcag cagcagcaatgt tcacttct cagaaagcctccggaaεctaaaaagccctcagcaccag agacagaagcagaεgga ccgεcc εεεaggagaεacaacagatgagcaaagaa gacag caagaggcaaaac cggacatagaggccaaccaacctεεggagaccaacaaagaaaaεε catccagtgtgac g a cagaccctgagacggaaaataaggcaggccagactccggaga acagcεcaεcaa ggccgagcεcctgaacga gεgccgε cacccεggccccgcaεg gc εggcagcacagggcacca cacεgacc cccgaccac εacεcccc a gatggcagcg aaaaccεatcacggεttεggεatgattccaccc εgaaaaεεcagεgc cεgεga tcaε aaccεεεaεg cεgtεεgcaccεεaacagcεεεaaaaεaεgcεcgccεa ε a cεaac ctgεεεgaεgcεctcεgccg εεcacεgεtεaaggtccεcagcaaggaεcacaaagcaaa gaaaa agcatεa g εcactacεcεa ttεaagaaaaaggεaca gεa acaaaεε gaactcaag tccac cccttcccccaεacaa aaata acaaac aggc atgaaggc t aggaaaggac cga ccεεcagaεggtc c caaaata aacaccεcaaaε acc εtagaagaacεgεgaaaaagaaεεgεggcaεεεεεcagεcacεacagcεccgaagεcεga gcaagaagεgggεgtgaagtc ccεc cεggε εgεaggaagεεgaaε agεgεεa cc εgcaεεεtt cεεccacagtgε aεgaatgaaεεcagaaaaaaag gccagtcagcε taatttc tcagaεgcat gaεgεggaatεεaaaacεcgεcc aaacagca gc ag tgcεgatacaaggatgcεagacctgctctgcgεgctc εεctggagεggcccaεεεcggε εctcεgaaacccacggcagccctttccaεcgεgaataaεcgt gεgεεcccaccεεεgtc ccctgccεtεtεgctactεcagεgcgcεctcεgaccagcε ccaaagaacεεtcccεgc t ctgcctcggttgccac gcεcεcct accacaεacgεεcccagεεεaεgaagaga c cacatt cctεεcaacccc ctgccεccCgaagaaaaacatcεcatgatga aca a a ttgcεgaεaacaccct atctagaaatεcgcεggccacaatacagaεggaaaεg c ac εgcεggaaaaacεgaaa caacccat ccaaεεagag aεaggcagaacacc agaεa gact εtagεtcεcgaaεaεccaεtaccεacεεεaaεgaaaacagaacεgccaεεggεca ataaacεgtaaagggaagaggtaaaεtgtgaεagagaaεt cεatgεcaεgggaaa εg aaaccaca aεε ga εaccagcaaεaεga εa aac c gεgccaagεεεεaag εaεatttεctcacaaagaεagagtgccatagtgaaacCaaacacεgεgcaεaaaggεaca εgaat acεccaguεt aaagεa atgcat g aa aaatgεggcaεgg a aεacaaacgcεacgεcact aaaagεcagaggaacaεtεcεaεεgacaaaaaεacgcεεc acεcacaεataaεgtcaccaεacggεgtcaatgatεaaaεtaga cεεcaca ag c acaaagcaεcagεctaggaaa atgacεta εactgaεgcaaatg gaacaεε gεagε agttttgεaaaagaacatcctttεcaaatatccacgtcaacgtacccccgaaattaaacj caagcacaεagεcagεεεgεctaaεtεtgεgεgaaaεεεεgεcgaaaaεaccεaaacaε;- ccatcεaεatεεgεgccεaccaεgεεεataaatgεtccaεaagεaccεεccaεgεεgtεc taεaaaaaaεg ataεεtaaaεaaatgtgaaεεaaaaεaaaaεεεεεgaεεaεεεεccg atccccg REFERENCES(Xi) SEQUENCE DESCRIPTION: SEQ ID NO.19 gccggaagtggggtg gaagccccggtgctggtgcggcgggggactgcggggccagccec aggtagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcagcag cagcagcaatgt tcacttct cagaaagcctccggaaεctaaaaagccctcagcaccag agacagaagcagaεgga ccgεcc εεεaggagaεacaacagatgagcaaagaa gacag caagaggcaaaac cggacatagaggccaaccaacctεεggagaccaacaaagaaaaεε catccagtgtgac ga cagaccctgagacggaaaataaggcaggccagactccggaga acagcεcaεcaa ggccgagcεcctgaacga gεgccgε cacccεggccccgcaεg gc εggcagcacagggcacca cacεgacc cccgaccac εacεcccc has gatggcagcg aaaaccεatcacggεttεggεatgattccaccc εgaaaaεεcagεgc cεgεga tcaε aaccεεεaεg cεgtεεgcaccεεaacagcεεεaaaaεaεgcεcgccεa ε has cεaac ctgεεεgaεgcεctcεgccg εεcacεgεtεaaggtccεcagcaaggaεcacaaagcaaa gaaaa agcatεa g εcactacεcεa ttεaagaaaaaggεaca gεa acaaaεε gaactcaag tccac cccttcccccaεacaa aaata acaaac aggc atgaaggc t aggaaaggac cga ccεεcagaεggtcac caaaaaageac ggcaεεεεεcagεcacεacagcεccgaagεcεga gcaagaagεgggεgtgaagtc ccεc cεggε εgεaggaagεεgaaε agεgεεa cc εgcaεεεtt cεεccacagtgε aεgaatgaaεεcagaaaaaaag gccagtcagcε taatttc tcagaεgcat gaεgεggaatεεaaaacεcgεcc aaacagca gc ag tgcεgatacaaggatgcεagacctgctctgcgεgctc εεctggagεggcccaεεεcggε εctcεgaaacccacggcagccctttccaεcgεgaataaεcgt gεgεεcccaccεεεgtc ccctgccεtεtεgctactεcagεgcgcεctcεgaccagcε ccaaagaacεεtcccεgc t ctgcctcggttgccac gcεcεcct accacaεacgεεcccagεεεaεgaagaga c cacatt cctεεcaacccc ctgccεccCgaagaaaaacatcεcatgatga aca aa ttgcεgaεaacaccct atctagaaatεcgcεggccacaatacagaεggaaaεg c ac εgcεggaaaaacεgaaa caacccat ccaaεεagag aεaggcagaacacc agaεa CTAG εtagεtcεcgaaεaεccaεtaccεacεεεaaεgaaaacagaacεgccaεεggεca ataaacεgtaaagggaagaggtaaaεtgtgaεagagaaεt cεatgεcaεgggaaa εg aaaccaca aεε ga εaccagcaaεaεga εa aac c gεgccaagεεεεaag εaεatttεctcacaaagaεagagtgccatagtgaaacCaaacacεgεgcaεa aaggεaca εgaat acεccaguεt aaagεa atgcat g aa aaatgεggcaεgg has aεacaaacgcεacgεcact aaaagεcagaggaacaεtεcεaεεgacaaaaaεacgcεεc acεcacaεataaεgtcaccaεacggεgtcaatgatεaaaεtaga cεεcaca ag c acaaagcaεcagεctaggaaa atgacεta εactgaεgcaaatg gaacaεε gεagε agttttgεaaaagaacatcctttεcaaatatccacgtcaacgtacccccgaaattaaacj caagcacaεagεcagεεεgεctaaεtεtgεgεgaaaεεεεgεcgaaaaεaccεaaacaε - ccatcεaεatεεgεgccεaccaεgεεεataaatgεtccaεaagεaccεεccaεgεεgtεc taεaaaaaaεg ataεεtaaaεaaatgtgaaεεaaaaεaaaaεεεεεgaεεaεεεεccg atccccg REFERENCES
1. Whilhem, P.J. Dynamic mutations hit double figures. Nature Genêt. 8, 213-215 (1994).1. Whilhem, P.J. Dynamic mutations hit double figures. Nature Broom. 8, 213-215 (1994).
2. Trottier, Y. et al. Cellular localization of the Huniington's disease protein and discrimination oî the normal and mutant form. Nature Genêt. 10, 104-110 ( 1995).2. Trottier, Y. et al. Cellular localization of the Huniington's disease protein and discrimination oî the normal and mutant form. Nature Broom. 10, 104-110 (1995).
3. Servadio, A. et al. Expression analysis of the ataxin- 1 protein in tissues from normal and spinocerebellar ataxia type 1 individuals.3. Servadio, A. et al. Expression analysis of the ataxin- 1 protein in tissues from normal and spinocerebellar ataxia type 1 individuals.
Narure Gêner. 10, 94-98 ( 1995).Embarrassing. 10, 94-98 (1995).
4. Yazawa, I. et al. Abnormal gène product identified in hereditary dentato-rubralpallidoluysian atrophy (DRPLA) brain. Nature Genêt. 10, 99-103 (1995). 5. Meier-Ewert, S., Maier, E, Ahmadi, A., Curtis, J. and Lebrach, H. An4. Yazawa, I. et al. Abnormal gene product identified in hereditary dentato-rubralpallidoluysian atrophy (DRPLA) brain. Nature Broom. 10, 99-103 (1995). 5. Meier-Ewert, S., Maier, E, Ahmadi, A., Curtis, J. and Lebrach, H. An
Automated approach to generating e pressed séquence catalogues. Nature 361, 375 (1993).Automated approach to generating e pressed sequence catalogs. Nature 361, 375 (1993).
6. Soares, M.B., Bonaldo, M.F., Jelene, P., Su, L, Lawton, L & Efstratiadis, A. Construction and characterization of a normalized cDNA library. Proc. Natl. Acad. Sci. USA. 91 , 9228-9232 ( 1994).6. Soares, M.B., Bonaldo, M.F., Jelene, P., Su, L, Lawton, L & Efstratiadis, A. Construction and characterization of a normalized cDNA library. Proc. Natl. Acad. Sci. USA. 91, 9228-9232 (1994).
7. Haad, D., Perry, M.J. and Haynes, L. Cellular transglutaminases in neural development. Int. J. Devl Neuroscience 1 1 , 709-720 ( 1993).7. Haad, D., Perry, M.J. and Haynes, L. Cellular transglutaminases in neural development. Int. J. Devl Neuroscience 11, 709-720 (1993).
8. Stott, K., Blackburn, J.M., Butler, P.J.G. & Perutz, M. Incorporation of glutamine repcats makes protein oligomerize. Implications for neuro- degenerative diseases. Proc. Natl. Acad. Sci. USA, 92, 6509-6513 ( 1995). 8. Stott, K., Blackburn, J.M., Butler, P.J.G. & Perutz, M. Incorporation of glutamine repcats makes protein oligomerize. Implications for neuro-degenerative diseases. Proc. Natl. Acad. Sci. USA, 92, 6509-6513 (1995).

Claims

REVENDICATIONS
1 ) Séquence d'ADN transcrit riche en triplet répété CAG ou CTG correspondant aux séquences A à I selon le tableau ainsi que l urs alleles normaux et mutés et les séquences complémentaires.1) Sequence of transcribed DNA rich in repeated CAG or CTG triplets corresponding to sequences A to I according to the table as well as their normal and mutated alleles and the complementary sequences.
2) Gène humain et ses alleles, caractérisé en ce qu'il porte au moins en partie une séquence selon la revendication 1.2) Human gene and its alleles, characterized in that it carries at least partially a sequence according to claim 1.
3) Procédé de mise en évidence des risques d'apparition d'une maladie à répétition trinucléotidique chez un patient, caractérisé en ce qu'on compare la séquence d'ADN selon la revendication 1 dudit patient à une séquence normale pour détecter la présence de répétitions trinucléotidiques supplémentaires.3) Method for highlighting the risks of the appearance of a trinucleotide recurrent disease in a patient, characterized in that the DNA sequence according to claim 1 of said patient is compared with a normal sequence to detect the presence of additional trinucleotide repeats.
4) Procédé selon la revendication 3, caractérisé en ce qu'on effectue une amplification de tout ou partie des séquences selon la revendication 1 et que l'on met en évidence dans le produit d'amplification la présence de répétitions trinucléotidiques supplémentaires.4) Method according to claim 3, characterized in that one carries out an amplification of all or part of the sequences according to claim 1 and that one highlights in the amplification product the presence of additional trinucleotide repeats.
5) Procédé selon la revendication 4, caractérisé en ce que l'amplification des séquences est effectuée avec les amorces décrites SEQ ID 1 à 18 correspondant aux séquences A à I respectivement. 6) Cellule transformée exprimant tout ou partie d'un gène selon la revendication 2.5) Method according to claim 4, characterized in that the amplification of the sequences is carried out with the primers described SEQ ID 1 to 18 corresponding to the sequences A to I respectively. 6) A transformed cell expressing all or part of a gene according to claim 2.
7) Protéine obtenue à partir d'une cellule transformée selon la revendication 6.7) Protein obtained from a transformed cell according to claim 6.
8) Protéine interagissant avec une protéi ne selon la revendication 7.8) Protein interacting with a protein according to claim 7.
9) Anticorps monoclonal correspondant à une protéine selon l'une des revendications 7 ou 8.9) Monoclonal antibody corresponding to a protein according to one of claims 7 or 8.
10) Vecteur exprimant une protéine selon l'une des revendications 7 ou 8. 1 1 ) Utilisation thérapeutique d'une protéine selon l'une des revendications 7 ou 8, d'un anticorps s n la revendication 9 ou d'un vecteur selon la revendication 10. 10) Vector expressing a protein according to one of claims 7 or 8. 1 1) Therapeutic use of a protein according to one of claims 7 or 8, an antibody sn claim 9 or a vector according to claim 10.
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WO2003014396A1 (en) * 2001-08-06 2003-02-20 Biomedlab Corporation Diagnosis method of multiplication disease of trinucleotide repeated sequence and a diagnosis kit

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WO1998056950A1 (en) * 1997-06-11 1998-12-17 Fondation Jean Dausset-Ceph Dna sequences rich in triplet repeat useful in the diagnosis of trinucleotide repeat disorders
WO2003014396A1 (en) * 2001-08-06 2003-02-20 Biomedlab Corporation Diagnosis method of multiplication disease of trinucleotide repeated sequence and a diagnosis kit

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CA2246362A1 (en) 1997-08-21
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